Copy number-dependent transgene expression and tamoxifen-dependent nuclear translocation of the CreERT2-fusion protein in neurons of the adult hippocampus. (A) The cassette encoding the CreERT2-fusion protein was inserted in frame at the ATG of the CaMKIIα gene present on a large DNA fragment of a BAC vector containing mouse genomic DNA by homologous recombination in bacteria. The genomic insert of the BAC vector contained a 43 kb 5'-upstream and a 100 kb 3'-downstream region of the CaMKIIα gene. (B-G) Immunohistochemical staining of pyramidal neurons in the hippocampal CA1 region of CaMKCreERT2 transgenic animals with different transgene copy numbers using an antibody against Cre recombinase revealed non-nuclear localization of the Cre recombinase in vehicle-injected animals (B, D, F) and nuclear localization in animals 12 hours after injection of 1 mg tamoxifen (C, E, G). The CaMKCreERT2 transgene copy number was determined by Southern blot analysis using a probe that detects restriction fragments of different sizes for the endogenous CaMKIIα gene (upper band) and the CaMKCreERT2 transgene (lower band) (H, I, J).